Fracture-label--a method that permits the cytochemical characterization of faces produced by freeze-fracture--was used to determine the partition and distribution of a glycolipid on membrane fracture-faces of Acanthamoeba castellanii cells. After treatment with Con A, the glycolipid--a lipophosphonoglycan (LPG)--was labeled with colloidal gold coated with horseradish peroxidase. The label was abundant over exoplasmic fracture-faces (face E) of plasma membranes but absent from protoplasmic fracture-faces (face P). We conclude that, in A. castellanii, glycolipid molecules are restrict to the outer half of the plasma membrane. This conclusion was confirmed by new experiments with cells disrupted by freezing and thawing, where access of label to the cell interior did not result in labeling of the inner surface. Our results are the first to establish the exclusive localization of a glycolipid to the outer half of a plasma membrane. Fracture-label is proposed as a new technique to investigate the distribution and partition of glycolipids in plasma and intracellular membrane halves.